Topochemistry of Environmentally Friendly Pretreatments To Enhance

Article pubs.acs.org/JAFC

Topochemistry of Environmentally Friendly Pretreatments To Enhance Enzymatic Hydrolysis of Sugar Cane Bagasse to Fermentable Sugar Hongyan Mou,*,† Elina Heikkila,̈ † and Pedro Fardim*,†,‡ †

Laboratory of Fibre and Cellulose Technology, Åbo Akademi University, Porthaninkatu 3, FI-20500 Turku, Finland Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, Jeddah 21589, Saudi Arabia

‡

ABSTRACT: In this work, dilute alkaline and alkaline peroxide pretreatments were conducted in comparison with hydrotropic pretreatment to improve the delignification of bagasse prior to enzymatic hydrolysis. The surface chemical composition of bagasse after pretreatments was investigated by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface distribution of lignin and extractives on the bagasse fiber was significantly changed by dilute alkaline, alkaline peroxide, and hydrotropic pretreatments. Hydrotropic pretreatment typically showed, other than the decrease of surface coverage by lignin and extractives, dramatic removal of xylan, thereby leading to more cellulose exposed on the fiber surface after pretreatment. Fiber morphology after pretreatments was more favorable for enzyme hydrolysis as well. However, the hydrotropic treatment had clear advantages because the enzymatic hydrolysis yields of glucan and xylan of pretreated bagasse were 83.9 and 14.3%, respectively. KEYWORDS: surface chemistry, hydrotropic pretreatment, enzymatic hydrolysis, sugar cane bagasse, XPS, ToF-SIMS

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INTRODUCTION Sugar cane bagasse is a sugar mill waste with low cost and excellent potential as feedstock for bioethanol production.1 Like other lignocellulosic materials, sugar cane bagasse consists of cellulose, hemicelluloses, lignin, and small amounts of extractives and has a low ash content compared to rice straw and wheat straw.2−4 From economic and environmental perspectives, sugar cane bagasse is more interesting as feedstock for bioethanol production than other nonwood raw materials. In past years, different pretreatment technologies such as liquid hot water, acid, alkaline, ionic liquids, and steam explosion have been introduced to treat sugar cane bagasse aiming to enhance the enzymatic hydrolysis through increasing the surface area, decreasing cellulose crystallinity, and degrading lignin.2,5−9 Alkaline pretreatment is an efficient chemical pretreatment for nonwood biomass. It is believed that lignin and some hemicelluloses are removed, therefore inducing cellulose swelling and changes of fiber morphology and, consequently, improving the enzyme hydrolysis yield.10,11 Alkaline peroxide is an environmentally friendly method used for removing lignin by the HOO• free radical formed during the process,12,13 and it has no detectable harmful byproducts for the fermentation stage such as furfural.7 Hydrotropic pretreatment is a process to extract lignin from lignocellulosic biomass using aqueous solutions of hydrotropes.14 The most typically used hydrotrope agent is sodium xylenesulfonate (SXS). The concentration of SXS must be at least 30% to solubilize the lignin from biomass into the water phase. In general, the treatment temperature can be 150−170 °C14−18 This method is more appropriate for hardwood delignification than softwood.18 Through controlling the treatment conditions, >90% of lignin in birch can be removed without generating sulfur bonds, and the lignin is easily precipitated after pretreatment.15 It was recently reported © 2014 American Chemical Society

that 30% hydrotrope agent solution was suitable for solubilizing the lignin from hardwood into the water phase at 150 °C for 2 h, leading to a glucan yield of birch of 84% after enzymatic hydrolysis.16 Also, hydrotropic pretreatment was suitable for nonwood biomass. With the addition of 0.17% acid, hydrotropic pretreatment was strengthened, lignin was efficiently removed both from the fiber cell wall and from the surface of the common reed, whereas xylan was significantly displaced, leading to the improvement of accessibility of enzyme to cellulose.17 Additionally, hydrotropic solvent is reusable and recyclable, which helps to control the process cost and save energy.14,18 So far, there is no ideal fractionation method to separate cellulose, hemicelluloses, and lignin from lignocellulosic biomass. Hydrotropic treatment has shown certain potential to be a cost-effective method.14,15 The distribution of lignin on the surface of biomass materials has shown to directly influence the enzyme hydrolysis efficiency.16 XPS and ToF-SIMS are surface sensitive techniques and able to supply the surface chemical composition and image the distribution of surface components in biomass and biomaterials.19−24 Most recently, XPS and ToF-SIMS were successfully used to monitor the effect of the hydrotropic treatment of biomass by assessing surface lignin and carbohydrates after pretreatment and after enzyme hydrolysis.16,17 In this work, hydrotropic pretreatment was used for the first time for sugar cane bagasse pretreatment. The advanced surface instruments XPS and ToF-SIMS were introduced for studying the various pretreatment and enzyme hydrolysis mechanisms of bagasse using a topochemistry Received: Revised: Accepted: Published: 3619

February 2, 2014 March 31, 2014 April 1, 2014 April 1, 2014 dx.doi.org/10.1021/jf500582w | J. Agric. Food Chem. 2014, 62, 3619−3625

Journal of Agricultural and Food Chemistry

Article

Table 1. Chemical Composition of Bagasse after Pretreatment glucan (%) Bref BN BH Bsxs a

40.9 56.1 54.9 77.3

(0.2)a (0.3) (0) (0.4)

xylan (%) 23.9 22.4 24.1 7.0

arabinan (%)

(0)a (0.1) (0.1) (0.01)

1.5 1.7 1.7 0.1

(0)a (0) (0) (0)

total lignin (%) 17.7 6.4 8.4 6.3

(3)a (0.1) (0.3) (1.1)

Rglucan (%)

Rxylan (%)

Rdelignification (%)

Ysolid (%)

83.4 (0.4)a 87.8 (0.1) 81.2 (0.6)

56.9 (0.3)a 66.2 (0.3) 12.6 (0.01)

78.0 (0.3)a 68.9 (1.1) 84.7 (2.7)

60.8 (2)a 65.5 (1) 43 (2)

The standard deviation is presented in parentheses.

perspective, and according to our knowledge hydrotropic pretreatment is an emerging technology.

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R glucan/xylan (%) =

MATERIALS AND METHODS

R delignification (%) = 1 −

Materials. Sugar cane bagasse was obtained from Centro de Tecnologia Canavieira, Brazil. For removal of sucrose, bagasse was washed with tap water until the washing liquor was colorless and thereafter stored in a freezer room. Sodium hydroxide (NaOH), 30% hydrogen peroxide (H2O2), and sodium xylene sulfonate (SXS) with 90% purity as hydrotropic solvent were purchased from Fluka. Commercial enzymes cellulase (Celluclast 1.5 L) and β-glycosidase (Novozyme 188) were kindly supplied by VTT Technical Research Centre of Finland. The activities of the cellulase and the β-glycosidase were 75 FPU/mL and 5900 nkat/mL, respectively. All chemicals and enzymes were used as received without further purification. Pretreatment Methods. Dilute Alkaline Pretreatment. Bagasse was treated by 0.2 g/g (substrate) NaOH at 60 °C in a water bath for 2 h. The liquid/solid ratio (w/w) was 10:1. After pretreatment, the residue of bagasse was washed until the pH was neutral.25 Sodium Hydroxide−Hydrogen Peroxide Pretreatment. Bagasse was treated by same dosage of NaOH as alkaline treatment together with 0.25 g/g (substrate) H2O2 in a 500 mL flask. The flask was put on a magnetic stirrer at 150 rpm for 24 h. To avoid decomposition of H2O2, the reaction was carried out in a dark place at room temperature. The liquor/wood ratio (w/w) was 10:1. Upon completion, the substrate was washed until neutral and then stored in a freezer.26 Hydrotropic Pretreatment. Bagasse was pretreated by 30% (w/v) SXS and 0.17% (w/v) formic acid in a revolving digester under the controlled temperature of 160 °C for 40 min; the liquor/wood ratio (w/w) was 10:1, and the initial pH was 3.5 ± 0.05. After pretreatment, the substrates were disintegrated and washed with 5% (w/w) NaOH solution and then washed by tap water until the washing liquor was colorless. After pretreatments, all treated samples were collected and weighed for further analysis and tests. Enzymatic Hydrolysis of Pretreated Samples. The pretreated samples were hydrolyzed by cellulase at a dosage of 20 FPU/g substrate together with 300 nkat β-glycosidase/g substrate at 50 °C in pH 5.0. Consistency was 20 mg/mL (w/v). Acetic acid buffer was used to modify the pH. The reaction time interval was 2, 6, 24, 48, and 72 h, respectively. The enzyme hydrolysis was ended by boiling for 10 min. After cooling with cold water, the hydrolysate was centrifuged at 1730g (3000 rpm) for 20 min. The supernatant was collected for sugar analysis. Chemical Composition Analysis. The reference sample and pretreated samples were first extracted with acetone for 5 h in a Soxhlet device. The extractive free samples were used for chemical composition analysis. Glucan, xylan, and lignin content of untreated and pretreated biomass were determined following the NREL procedure for the determination of structural carbohydrates and lignin in biomass.27 The loading yield after pretreatments (Ysolid), the recovered ratio of glucan or xylan (Rglucan/xylan), and the delignification rate (Rdelignification) were calculated by the following formulas:

Ysolid (%) =

pretreated biomass (g) × 100 original biomass (g)

Ysolid × Cglucan/xylan of pretreated biomass Cglucan/xylan of original biomass

(2)

Ysolid × C lignin of pretreated biomass C lignin of original biomass

(3)

Glucan and xylan of both enzyme and sulfuric acid hydrolyzed pretreated samples (45 μm syringe film filtered) were detected by using a HPLC system (model 1200, Agilent, USA) equipped with a Bio-Rad Aminex HPX-87H column (300 mm × 7.8 mm) and refractive index detector. The column was operated at 55 °C with 0.005 mol/L H2SO4 solution as the mobile phase at a flow rate of 0.5 mL/min. Sample injection volume was 20 μL. Quantitative analysis was performed using a calibration with external standards of known concentrations. For each experiment on the pretreatments and the enzymatic hydrolysis, at least three replicates were made, and the average of the results was reported. The glucan and xylan yield after enzymatic hydrolysis was calculated on the basis of the glucan and xylan contents of the initial raw material. Topochemical Characterization. Untreated and treated biomasses were evaluated with field emission scanning electron microscope (FE-SEM) LEO 1530 Gemini equipment. Samples were previously coated with carbon using a coating system equipped with a rotating base. Images were obtained with 500× and 15000 × magnifications. X-ray spectroscopy (XPS) spectra were obtained with a Physical Electronics PHI Quantum 2000 ESCA instrument equipped with a monochromatic Al Kα X-ray source. Low-resolution spectra were measured in 3.5 min with a pass energy of 187 eV and high-resolution spectra of C1s peak in 9 min using a pass energy of 23 eV. At least four different spots were measured on each sample. The O/C ratios were calculated from the low-resolution XPS spectra. Both acetoneextracted samples (Soxhlet, acetone/water mixture 9:1 (v/v), overnight) and unextracted samples after freeze-drying were analyzed. The surface coverage by lignin (Slig), carbohydrates (Scarb), and extractives (Sext) were calculated according to the following equations using the average O/C ratio value:17,19,24

Sext = (O/Cextracted − O/C before extraction) /(O/Cextracted − O/Cextractives) × 100

(4)

S lig = (O/Cextracted − O/Ccarbohydrate) /(O/C lignin − O/Ccarbohydrate) × 100

Scarb = 100 − S lig % O/Ccarbohydrate = 0.83,

(5) (6)

O/C lignin = 0.33,

O/Cextractives = 0.09 ATR-FTIR was used to analyze the samples after pretreatment. Small amounts of freeze-dried samples after pretreatment were placed on the diamond probe of Nicolet IS 50 FTIR (software OMNIC 7.3, wavenumbers in cm−1). Thirty-six scans were acquired for each sample, recorded from 4000 to 400 cm−1. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed with a Physical Electronics ToF-SIMS TRIFT II spectrometer on the pretreated and enzymatically

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dx.doi.org/10.1021/jf500582w | J. Agric. Food Chem. 2014, 62, 3619−3625


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hydrolyzed samples. All of the samples were freeze-dried after the acetone extraction. The instrument was equipped with a primary ion beam of 69Ga+ liquid metal ion source and an electron flood gun for charge compensation. Positive secondary ions were detected using 25 kV acceleration voltage in 8 min. At least three measurements were done on each sample.

before and after sample extraction are given. As can be seen, the removal of carbon-rich material from the bagasse surface during pretreatments and, on the other hand, during the acetone extraction, is demonstrated by the increase of the O/C ratio. Furthermore, the surface coverage by lignin, carbohydrates, and extractives of bagasse after different pretreatment methods was gained from the XPS results (Table 2). After the pretreatments applied, the surface of bagasse samples contained more carbohydrates and less lignin than the reference sample. The surface coverage by lignin value of BN and Bsxs were on the same level. However, during alkaline pretreatment, together with lignin removal a notable amount of extractives was reduced from the fiber surface as well. Table 2 also shows that the O/C value of BH after extraction was close to that of pure cellulose. However, this high O/C ratio was probably caused by oxidation of surface lignin and insertion of oxygen atoms to form carbonyl and carboxyl groups. According to the results shown in Table 1, the residual total lignin of BH was 8.4%, which was higher than that of BN and Bsxs, but the surface coverage by lignin for BN and Bsxs was >11%, whereas the BH had 0%. This was a limitation of the XPS method to assess detailed chemical changes in surface lignin. ATR-FTIR and ToF-SIMS spectrometry methods were applied here to complement XPS data. ATR-FTIR of Bagasse after Pretreatment. The samples after pretreatment were measured by ATR-FTIR. The analysis depth of ATR-FTIR was from 1 μm, which was deeper than XPS (5−10 nm). The spectra are shown in Figure 1, and the bonding vibrations are summarized in Table

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RESULTS AND DISCUSSION Chemical Composition of Bagasse after Pretreatments. The chemical compositions of bagasse after dilute alkaline, alkaline peroxide, and hydrotropic pretreatments are given in Table 1. With dilute alkaline, alkaline peroxide, and hydrotropic pretreatments, the total amount of lignin in bagasse was reduced from the original 17.7 to 6.4, 8.4, and 6.3%, respectively. The pretreatment technology was applied before enzymatic hydrolysis process to overcome the limits for enzyme access to the polysaccharides, where cell wall lignin is one of the main causes of recalcitrance.16,17 Lignin was removed as expected by all three of the used pretreatment methods, typically by hydrotropic pretreatment. Another significant difference was detected between the methods with regard to their impact on the bagasse carbohydrates. As shown in Table 1, hemicelluloses were less degraded by diluted alkaline and alkaline peroxide pretreatment than by hydrotropic pretreatment. However, hydrotropic pretreatment could remove both lignin and hemicelluloses. For instance, arabinan was almost totally degraded, and xylan was efficiently removed, whereas only slightly lower glucan was recovered in the hydrotropic process compared with other methods used (Table 1). As reported, acid pretreatment and steam explosion could also remove hemicelluloses, but acid pretreatment may cause equipment corrosion,6 whereas steam explosion may require high capital cost, particularly for large-scale production.8 Although alkali-based pretreatment can remove lignin, high capital cost in the chemical recovery system or wastewater treatment may be needed. Therefore, hydrotropic pretreatment was appropriate for removing xylose and lignin from bagasse, and it was an efficient technology to remove mainly hemicellulose in comparison with acid pretreatment and steam explosion. Certainly, more extensive studies of hydrotropic pretreatment (e.g., the optimization of pretreatment conditions, economic evaluation for overall process) for different feedstocks should be performed in the future. Furthermore, the recovery of lignin and xylan from hydrotropic pretreatment process can be a promising biorefinery application for producing high-value-added chemicals and biomaterials.14,15 Surface Chemical Composition after Pretreatment Analyzed by XPS. The elemental composition of the sample surfaces was investigated by XPS. The main constituents were carbon and oxygen. In the reference sample (Bref), additionally small amounts of N, Al, and Si were detected in the XPS lowresolution spectra. These were probably originated from the native constituents in sugar cane. In Table 2, the O/C ratios

Figure 1. ATR-IR of bagasse after pretreatment. Bref, reference sample of sugar cane bagasse; Bsxs, hydrotropic pretreated sample; BN, alkaline pretreated sample; BH, alkaline peroxide pretreated sample.

3.5,10,25−32 The broad band from 3340 cm−1 has been reported as the O−H bond that could be found in carbohydrates and lignin, and for agriculture materials, the signal also possibly arises from the aliphatic fraction of waxes.5,10 With the different pretreatment methods, this group did not obviously change. In Figure 1, 2896 cm−1 is the CH bond in polysaccharides.30 It was stronger in the pretreated bagasse samples. The signal at 1733 cm−1 is attributed to the CO of acetyl groups in hemicellulose that was obviously shown in the untreated bagasse but not detected in BN, BH, or Bsxs samples, because of the removal of acetyl groups during pretreatment.30 The

Table 2. Surface Chemical Composition of Bagasse after Pretreatment As Detected by XPS sample Bref BN BH Bsxs a

O/Cext 0.68 0.77 0.84 0.77

(0.11)a (0.06) (0.11) (0.04)

O/C 0.45 0.69 0.63 0.61

(0.03)a (0.03) (0.03) (0.02)

Sexts (%)

Slig (%)

Scarb (%)

38.1 12.6 28.6 24.7

31.0 11.8 0 11.3

69.0 89.2 100 89.7

The standard deviation is presented in parentheses. 3621



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Table 3. Chemical Bond Assignment of ATR-FTIR for Bagasse after Pretreatments wavenumber (cm−1) 3340 2896 1730 1605 1510 1240 1036 904 835

vibration

source

OH stretching water/polysaccharides CH (CH2) stretching polysaccharides CO stretching acetyl groups of hemicelluloses CPh aromatic ring lignin CC aromatic ring lignin CO guaiacyl lignin COC polysaccharides CH deformation lignin CH out of plane p-coumaric ester groups

signals at 1605 and 1510 cm−1 were signs of lignin, and 1240 cm−1 was assigned to the aryl C−O bond from guaiacyl lignin, which existed only in untreated bagasse, indicating lignin dissolution during pretreatment.28,31 The signal at 904 cm−1 was assigned as the β-glucosidic bond, which was stronger in the pretreated samples because of more carbohydrate on the fiber surface,28 and this is in agreement with the results by XPS in Table 2. The signal at 835 cm−1 has been reported as the ester carbonyl signal of p-coumaric ester groups,31 and it was detected only in the untreated sample. Surface Morphology after Pretreatment. The surface morphology of bagasse samples after pretreatment was investigated by FE-SEM. In Figure 2a, the reference bagasse fiber was covered by some fragments, probably fibrils created by mechanical forces during the sugar cane squeeze stage in the sugar mill. The pits in the cell wall were shown in all samples at such high magnification, 15000×. Shrinkage trace was present on alkaline-treated fiber that proved the fiber swelling caused by alkaline pretreatment in Figure 2b. Alkaline treatment could swell wheat straw fiber, leading to the increase of the internal surface,11 and the same action to sugar cane bagasse was found. After alkaline peroxide pretreatment, some substances formed spider web-like structures covering the fiber surface in Figure 2c. A similar phenomenon was found on softwood fiber surface after mechanical refining treatment because of xylan released from the fiber wall readsorbing on the surface.16 It was probably due to the high surface carbohydrates covering bagasse fiber, as detected by XPS (Table 2). These substances were probably released from the collapsed inner fiber wall, which is shown in Figure 2d. After hydrotropic pretreatment, the fibrils could be seen in Figure 2e. It seems that the surface cell wall was ripped off. Fibers were expanded and more pores exposed. The performance of the fiber digestibility improved by pretreatment cannot be sufficiently explained by the change of chemical composition only. The fiber structure features and cell wall damage created by the pretreatment procedure could assist the enhancement of the fiber accessibility for enzyme as well.32,33 The factors affecting the fiber accessibility are complementing each other. Enzymatic Hydrolysis of Pretreated Bagasse. After dilute alkaline, alkaline peroxide, and hydrotropic pretreatments, the glucan and xylan yields of bagasse were studied (Figures 3 and 4). The maximum glucan yields of bagasse samples BN, BH, and Bsxs were 78.3, 73, and 83.9%, respectively. The glucan yield achieved from low-temperature alkaline pretreatment at 60 °C for 2 h was better than that from the sodium hydroxide peroxide pretreatment at room temperature for 24 h. This was because of the lesser contents of lignin

Figure 2. Morphology of bagasse after pretreatment (Bref (a), BN (b), BH(c, d), Bsxs (e)).

Figure 3. Glucan yield of bagasse after enzyme hydrolysis.

and hemicelluloses in BN (Table 1), as both lignin and hemicelluloses are key barriers for glucan conversion.34 On the other hand, the surface distribution of lignin and xylan is also of critical importance for enzymatic hydrolysis.17 According to the results in Figure 3, the highest glucan yield could be achieved 3622



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results obtained, it could be concluded that among the studied pretreatment techniques, the hydrotropic method was the most efficient one to significantly improve the glucan conversion for bagasse. Surface Chemical Characterization by ToF-SIMS. The surface chemical composition and the component distribution on the outermost surface of bagasse after alkaline, sodium hydrogen peroxide, and hydrotropic pretreatments were studied by TOF-SIMS. Samples after the following enzymatic hydrolysis were investigated also. The surface sensitivity and specificity of ToF-SIMS is 1−3 nm. Thus, ToF-SIMS can provide more detailed information on surface chemical compositions.16,35 Sugar monomers from hexosans (cellulose, mannan, galactan) were identified by ToF-SIMS spectrum at m/z 127 and 145 and from pentosans at m/z 115 and 133. In ToF-SIMS, lignin gives the characteristic mass fragments m/z 107 and 121 from the p-hydroxyphenyl (H) unit, m/z 137 and 151 from the guaiacyl (G) unit, m/z 167 and 181 from the syringyl (S) unit,36 and m/z 77 and 91 from the general aromatic ring.21,37 In Figure 5, the mass region m/z 100−200 of the alkaline hydrogen peroxide and hydrotropic pretreated samples is displayed as well as the same after enzymatic hydrolysis. In general, in the pretreated samples the sugar monomer fragments were clearly seen in the spectra. After the enzymatic hydrolysis again, the lignin peaks were visually more evident in

Figure 4. Xylan yield of bagasse after enzyme hydrolysis.

by hydrotropic pretreatment. This is due to the lower lignin and xylan content (Table 1) as well as the lesser surface coverage by lignin and xylan (Table 2) on bagasse fibers after hydrotropic pretreatment. Similar results were also reported for the pretreatment of common reed.17 The low xylan yield for the case of hydrotropic pretreatment (Figure 4) was because of the low xylan recovery (Table 1). Therefore, on the basis of the

Figure 5. Positive ion ToF-SIMS spectra of BH, EBH, Bsxs, and EBsxs in the mass range m/z 100−200. 3623



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was significantly removed. The surface morphology features after hydrotropic pretreatment also benefit enzyme access to fibers. A new biorefinery platform combining production of cellulosic ethanol and recovery of lignin and xylan can be developed on the basis of our results.

the spectra. This was verified when the peak intensities were compared numerically. The relative peak intensities of the characteristic mass peaks of carbohydrates and lignin were calculated (Table 4). The

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Table 4. Ratio of the Peak Intensities of the Characteristic Mass Peaks of Carbohydrates and Lignin between Bagasse (B) Samples after Pretreatment and Enzymatic Hydrolysis (E) in ToF-SIMS sample Bref BN BH Bsxs EBref EBN EBH EBsxs a

carbohydrates/lignin 0.67 1.09 1.27 1.82 1.02 0.36 0.47 0.48

(0.07)a (0.51) (0.48) (0.43) (0.27) (0.01) (0.04) (0.12)

hexose/total × 10

3

1.1 2.3 2.6 4.8 3.1 0.7 0.6 0.8

pentose/total × 10

(0.3)a (1.4) (1.1) (1.3) (0.7) (0.1) (0.3) (0.3)

1.7 3.1 3.1 3.7 2.7 1.2 1.0 2.2

AUTHOR INFORMATION

Corresponding Authors

*(H.M.) E-mail: hmou@abo.fi. *(P.F.) E-mail: pfardim@abo.fi,

3

Notes

(0.6)a (1.8) (1.8) (0.7) (0.2) (0.1) (0.4) (0.9)

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Top Analytica Ltd. for providing surface analytical instruments.The enzymes were kindly supplied from VTT Technical Research Centre of Finland.

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ABBREVIATIONS USED Bref, sugar cane bagasse reference sample; BH, alkaline peroxide pretreated sugar cane bagasse sample; BN, alkaline pretreated sugar cane bagasse sample; Bsxs, hydrotropic pretreated bagasse sample; SXS, sodium xylenesulfonate; XPS, X-ray electron xpectroscopy; ATR-FTIR, attenuated total reflectance-infrared spectra; ToF-SIMS, time of flight secondary ion mass spectrometry; FE-SEM, field emission-scanning electron microscope; Sext, surface coverage by extractives; Slig, surface coverage by lignin; Scarb , surface coverage by carbohydrates

The standard deviation is presented in parentheses.

bagasse samples after both pretreatment and enzyme hydrolysis were studied in Table 4. The increasing ratio between carbohydrate and lignin peak intensities reflects lignin reduction on the fiber surface. After hydrotropic pretreatment, the ratio value between carbohydrates and lignin was increased about 3 times compared with the reference. This supports the finding that lignin on the surface was profusely removed by hydrotropic pretreatment (Table 2). The peak intensities of hexose monomer fragments and pentose monomer fragments in the ToF-SIMS spectra were also compared (Table 4). For that, the peak intensities were normalized individually by the total counts in the spectrum. After the hydrotropic pretreatment, the ratio value of hexose to total was raised to 4 times compared to the reference. Hexose of bagasse is mainly from cellulose that contains glucose according to the results shown in Table 1. Results from ToF-SIMS are in good agreement with XPS in Table 2. After enzymatic hydrolysis, the ratio value of carbohydrates to lignin was decreased following the hydrolyzation and removal of carbohydrates from the sample surfaces. This was not detected in the reference sample. It can be seen as a clear indication of the impact of the pretreatments on the hydrolysis efficiency by reducing the biomass recalcitrance. The value had no large difference between the different pretreatment methods. Hexose and pentose ratios follow the same trend. Especially for the hydrotropic pretreated bagasse, the hexose ratio was reduced from 4.8 to 0.8. Most of the glucose was hydrolyzed by hydrotropic pretreatment. In the case of BH and BN, xylan hydrolysis was also contributing to the decreasing ratio between carbohydrates and lignin. Thus, ToF-SIMS also demonstrated the high efficiency of hydrotropic pretreatment on delignification and enzyme saccharification. Overall, the bulk chemical composition, surface chemical distribution, and fiber morphology are correlated and should be considered together for comprehensive understanding of the mechanism of pretreatment to accelerate enzymatic hydrolysis. The glucan yield of sugar cane bagasse by enzymatic hydrolysis was advanced by mildly alkaline and alkaline hydroxide pretreatments at room temperature. A much better improvement was achieved with hydrotropic pretreatment through lignin removal and change in surface distribution. Typically for hydrotropic pretreatment, in addition to delignification, xylan

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Topochemistry of Environmentally Friendly Pretreatments To Enhance

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